CN113684220A - Single gene hypertension gene detection kit based on mutated VHL gene - Google Patents

Single gene hypertension gene detection kit based on mutated VHL gene Download PDF

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CN113684220A
CN113684220A CN202111038660.5A CN202111038660A CN113684220A CN 113684220 A CN113684220 A CN 113684220A CN 202111038660 A CN202111038660 A CN 202111038660A CN 113684220 A CN113684220 A CN 113684220A
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刘哲
梁庆渊
赵娜娜
赖开生
刘昕超
高璇
李方玉
侯青
惠汝太
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Bestnovo Beijing Medical Technology Co Ltd
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Abstract

The invention relates to the technical field of human genetics and internal medicine cardiovascular, in particular to a mutated VHL gene, wherein a base T is mutated into a base G at a genomic position chr3: 10191647; the reference genomic version is GRCh 37. The invention also relates to a kit for detecting the single-gene hypertension gene, which comprises a primer for amplifying the mutated VHL gene. The mutated VHL gene provided by the invention can be used as a biomarker for clinical auxiliary diagnosis; the carrier for detecting the variation provides a bearing guide and genetic counseling for the prenatal and postnatal care of a subject, reduces the birth of a child patient, and has important significance for early diagnosis of pheochromocytoma in monogenic hypertension or auxiliary clinical judgment.

Description

Single gene hypertension gene detection kit based on mutated VHL gene
Technical Field
The invention relates to the technical field of human genetics and internal medicine cardiovascular, in particular to a single gene hypertension gene detection kit based on a mutated VHL gene.
Background
The monogenic hypertension belongs to secondary hypertension, is monogenic hereditary diseases taking cardiovascular damage as a unique phenotype or accompanied with the cardiovascular damage, and has at least 19 pathogenic genes, 9 genes causing pheochromocytoma and 10 pathogenic genes causing salt-sensitive hypertension. Among them, pheochromocytoma is derived from neural crest, belongs to the tumor of pheochromocytoma tissue of adrenergic system, mainly occurs in suprarenal medulla and sympathetic nervous system, and is benign, and malignant pheochromocytoma accounts for 7-15%, and is a potentially dangerous tumor.
With the continuous development of gene detection technology, the susceptible genes related to the origin and development of pheochromocytoma are VHL, NF1, RET, SDHB and SDHD, wherein the VHL gene is listed as the first choice factor for genetic pheochromocytoma mutation screening as the gene for researching the earliest mutation and the most detected mutation of the pheochromocytoma.
The protein encoded by the VHL gene comprises protein complex components of elongatin B, elongatin C and cullin-2, and has ubiquitin ligase E3 activity. The protein is involved in ubiquitination and degradation of Hypoxia Inducible Factor (HIF), a transcription factor that plays a central role in oxygen regulation gene expression. RNA polymerase II subunit POLR2G/RPB7 has also been reported as the target of this protein. VHL gene mutations are associated with the development of familial pheochromocytoma with autosomal dominant inheritance, as well as with the development of Von Hippel-Lindau syndrome (VHL disease) with autosomal dominant inheritance and familial polycythemia 2 with autosomal recessive inheritance.
Although many VHL mutation sites related to pheochromocytoma are found at present, a large number of unknown mutation sites still exist, and further finding out a new VHL gene mutation site is helpful for further researching the pheochromocytoma in single-gene hypertension, and has important significance for early diagnosis of the pheochromocytoma or assistance of clinical judgment.
Disclosure of Invention
The invention aims to provide a single-gene hypertension gene detection kit based on a mutated VHL gene aiming at pheochromocytoma in single-gene hypertension.
The technical scheme provided by the invention is as follows:
the invention provides a mutated VHL gene, wherein at the genomic position chr3:10191647, a base T is mutated into a base G; the reference genomic version is GRCh 37.
Preferably, the sequence of the mutated VHL gene at genomic positions chr3:10191630-3:10191681 is SEQ ID NO 2.
The invention also provides a single-gene hypertension gene detection kit which comprises a primer for amplifying the mutated VHL gene.
Preferably, the sequence of the primers is SEQ ID NO. 7 and SEQ ID NO. 8.
Thirdly, the principle and the beneficial effects of the invention are as follows:
the mutated VHL gene disclosed by the invention can be used as a biomarker for clinically and auxiliarily diagnosing pheochromocytoma, and has important significance for early diagnosis or auxiliary clinical judgment; the detection kit developed based on the mutated VHL gene can detect the patient with the mutated VHL gene, can guide genetic block, provides a bearing guide and genetic counseling for a subject, and reduces the birth of the infant.
Drawings
FIG. 1 is a family diagram of example 1;
FIG. 2 is a Sanger sequencing chart of the proband in example 1;
fig. 3 is a Sanger sequencing graph of the father of the proband of example 1.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1 proband verification experiment
Sample source: in the children hospital in Hebei province, 5-10mL of whole blood samples are sent under the premise that a proband (female, 8 years old) and family members voluntarily sign informed consent, a medical record database is established, and the data of the disease condition, family condition and the like of the proband are recorded in detail. The study was approved by the ethical committee of the unit.
Description of the prior patient history:
TABLE 1 Procedent medical history description
Figure BDA0003248371110000021
Figure BDA0003248371110000031
Carrying out gene detection on VHL genes of probands and families thereof by adopting a Sanger sequencing method, and specifically comprising the following steps:
s1, extracting genome DNA:
the whole genome DNA extraction reagent of the magnetic bead method whole genome DNA extraction reagent of Jiangsu Baishinuo medical science and technology Limited company is adopted to extract the whole genome DNA of the anticoagulation sample of the human whole blood EDTA of the proband and the family members thereof, and the concentration and the purity of the DNA are detected.
S2, preparing a PCR reaction system:
design of specific amplification primers containing the fragment of interest:
upstream primer (VHL-E2-P-F2, SEQ ID NO: 7): 5'TACTACAGAGGCATGAACACC 3';
downstream primer (VHL-E2-P-R2, SEQ ID NO: 8): 5'CAGACAAGTCACCAACCGCTA 3';
length: 792 bp.
As the amplification reagent, 2 XTAQA MasterMix (Dye) produced by Jiangsukang, a century Biotech Co., Ltd was used.
An amplification system: 2 × Taq Master Mix (Dye)25 μ L; 1. mu.L of each of the upstream and downstream primers (10. mu.M); DNA template<0.5 mu g; by ddH2And O is supplemented to 50 mu L.
PCR amplification conditions: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles. Final extension at 72 ℃ for 2 min.
And (3) product detection: taking 2 mu L of PCR product, detecting the PCR product by using 1.5% agarose gel electrophoresis, selecting 1000bp Marker as reference, and detecting and verifying that the amplification product is the expected size.
S3, PCR products were sequenced using a 3730XL Genetic Analyzer full-automatic sequencer. The reference sequences and sequencing results were obtained from the NCBI (https:// www.ncbi.nlm.nih.gov /) database and aligned.
The experimental results are as follows:
(1) in this example, it was found that proband carries a mutated VHL gene, and the specific detection results are shown in table 2 below:
TABLE 2 specific test results for mutated VHL genes
Gene Genomic position Transcript number Base change Amino acid changes Reference genome version
VHL chr3:10191647 NM_198156 c.517T>G p.Ter173GlyextTer14 GRCh37/hg19
At genomic positions chr3:10191630-chr3:10191681, the sequence of the wild-type VHL gene is SEQ ID NO: 1: CAACGGATGGGAGATTGAagatttctgttgaaacttacactgtttcatct，TIs genome chr3 pre-mutation base at 10191647;
at genomic position chr3:10191630-3:10191681, the sequence of the mutated VHL gene is SEQ ID NO: 2: CAACGGATGGGAGATGGAagatttctgttgaaacttacactgtttcatct，GIs the mutated base at the gene group chr3: 10191647.
The reference sequence of the wild-type VHL gene encoding DNA is SEQ ID NO: 3:
atgccccggagggcggagaactgggacgaggccgaggtaggcgcggaggaggcaggcgtcgaagagtacggccctgaagaagacggcggggaggagtcgggcgccgaggagtccggcccggaagagtccggcccggaggaactgggcgccgaggaggagatggaggccgggcggccgcggcccgtgctgcgctcggtgaactcgcgcgagccctcccaggtcatcttctgcaatcgcagtccgcgcgtcgtgctgcccgtatggctcaacttcgacggcgagccgcagccctacccaacgctgccgcctggcacgggccgccgcatccacagctaccgagtgtatactctgaaagagcgatgcctccaggttgtccggagcctagtcaagcctgagaattacaggagactggacatcgtcaggtcgctctacgaagatctggaagaccacccaaatgtgcagaaagacctggagcggctgacacaggagcgcattgcacatcaacggatgggagattga，tis the base before mutation at position 517 of a reference sequence of the coding DNA.
519 base sequences in the DNA sequence coding for the mutated VHL gene are SEQ ID NO 4:
atgccccggagggcggagaactgggacgaggccgaggtaggcgcggaggaggcaggcgtcgaagagtacggccctgaagaagacggcggggaggagtcgggcgccgaggagtccggcccggaagagtccggcccggaggaactgggcgccgaggaggagatggaggccgggcggccgcggcccgtgctgcgctcggtgaactcgcgcgagccctcccaggtcatcttctgcaatcgcagtccgcgcgtcgtgctgcccgtatggctcaacttcgacggcgagccgcagccctacccaacgctgccgcctggcacgggccgccgcatccacagctaccgagtgtatactctgaaagagcgatgcctccaggttgtccggagcctagtcaagcctgagaattacaggagactggacatcgtcaggtcgctctacgaagatctggaagaccacccaaatgtgcagaaagacctggagcggctgacacaggagcgcattgcacatcaacggatgggagatgga，gis a mutated base.
c.517t > G represents: the base T at position 517 is mutated to a base G as compared to a reference sequence of a wild-type VHL gene encoding DNA.
The wild VHL gene coding protein is SEQ ID NO: 5:
MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRVYTLKERCLQVVRSLVK PENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD。
the 173 proteins encoded by the mutated VHL gene are SEQ ID NO: 6:
MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRVYTLKERCLQVVRSLVK PENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGDGthe 173 th stop codon was changed to nonpolar G (glycine, Gly).
p. ter173glyxtter14 denotes: the stop codon at position 173 was changed to nonpolar glycine (Gly, G) and the VHL protein was increased by 14 amino acids.
The mutation was found to be a rare mutation by querying the population frequency database (thousand genomes: none, ESP 6500: none, ExAC: none). This variation changes the 173 th stop codon to a nonpolar glycine, potentially resulting in prolonged expression of the protein, adding 14 amino acids to the VHL protein. The ClinVar and HGMD databases are queried to find that the variation is not found, and the ClinVar database is queried to find that other variations at the site, namely c.518G > T (p.Ter173Leu), c.519A > T (p.Ter173CysextTer15) and c.519A > G (p.Ter173TrpextX), are rated as pathogenic variations by a reporter, which indicates that the site is possibly a mutation hotspot of VHL syndrome. The literature search does not find the mutation and disease related report. According to the existing evidence: the variation is a rare variation, which may lead to prolonged expression of the protein, and the site is a mutational hot spot of VHL syndrome, which is a highly suspicious pathogenic mutation.
(2) FIG. 1 is a family diagram of example 1; FIG. 2 is a Sanger sequencing chart of the proband in example 1; fig. 3 is a Sanger sequencing graph of the father of the proband of example 1.
As shown in fig. 1 to 3, the mutant VHL gene carried by proband is inherited from proband mother, and the mutant VHL gene of proband mother is inherited from proband father. In addition, in this family, probangjiu and probabi nephew all carry the heterozygous missense variation of the VHL gene c.517T > G.
Example 2 Single Gene hypertension Gene detection kit
This example provides a kit for detecting c.517T > G heterozygous missense variation of human VHL gene, comprising 2 XTAQA MasterMix (Dye), primers capable of detecting mutated VHL gene, etc., the specific composition of the kit is shown in Table 3 below.
The specific steps of screening the patients with c.517T > G VHL gene mutation by using the kit are as follows: the DNA of the subject was extracted according to the procedure of example 1, then the VHL gene was amplified using the designed primer combination (SEQ ID NO:7 and SEQ ID NO:8) to obtain a PCR product, and finally the PCR product was sequenced.
Obtaining a reference sequence from an NCBI (https:// www.ncbi.nlm.nih.gov /) database, comparing the reference sequence with a sequencing result, judging whether the VHL gene of a tested person carries c.517T > G heterozygosis variation, and assisting the clinical confirmation of whether the tested person has a patient with c.517T > G VHL gene mutation.
TABLE 3 kit composition
Figure BDA0003248371110000061
Example 3 mutation verification against non-familial normal persons
The mutation site of VHL gene c.517T > G was detected in 984 ethnic groups of unrelated normal persons (i.e., out-of-family normal persons) by the method of example 1 or by the kit for detecting single gene hypertension gene of example 2, and none of the mutations could be detected.
The results show that c.517t > G heterozygous missense variations based on the VHL gene resulted in alterations of the protein encoded by the VHL gene, which is a known pathogenic gene, thus again demonstrating that c.517t > G heterozygous missense variations of the VHL gene are pathogenic mutations.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Baishinuo (Beijing) medical science and technology Co., Ltd
<120> detection kit for single gene hypertension gene based on mutant VHL gene
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> homo sapiens
<400> 1
caacggatgg gagattgaag atttctgttg aaacttacac tgtttcatct 50
<210> 2
<211> 50
<212> DNA
<213> homo sapiens
<400> 2
caacggatgg gagatggaag atttctgttg aaacttacac tgtttcatct 50
<210> 3
<211> 519
<212> DNA
<213> homo sapiens
<400> 3
atgccccgga gggcggagaa ctgggacgag gccgaggtag gcgcggagga ggcaggcgtc 60
gaagagtacg gccctgaaga agacggcggg gaggagtcgg gcgccgagga gtccggcccg 120
gaagagtccg gcccggagga actgggcgcc gaggaggaga tggaggccgg gcggccgcgg 180
cccgtgctgc gctcggtgaa ctcgcgcgag ccctcccagg tcatcttctg caatcgcagt 240
ccgcgcgtcg tgctgcccgt atggctcaac ttcgacggcg agccgcagcc ctacccaacg 300
ctgccgcctg gcacgggccg ccgcatccac agctaccgag tgtatactct gaaagagcga 360
tgcctccagg ttgtccggag cctagtcaag cctgagaatt acaggagact ggacatcgtc 420
aggtcgctct acgaagatct ggaagaccac ccaaatgtgc agaaagacct ggagcggctg 480
acacaggagc gcattgcaca tcaacggatg ggagattga 519
<210> 4
<211> 519
<212> DNA
<213> homo sapiens
<400> 4
atgccccgga gggcggagaa ctgggacgag gccgaggtag gcgcggagga ggcaggcgtc 60
gaagagtacg gccctgaaga agacggcggg gaggagtcgg gcgccgagga gtccggcccg 120
gaagagtccg gcccggagga actgggcgcc gaggaggaga tggaggccgg gcggccgcgg 180
cccgtgctgc gctcggtgaa ctcgcgcgag ccctcccagg tcatcttctg caatcgcagt 240
ccgcgcgtcg tgctgcccgt atggctcaac ttcgacggcg agccgcagcc ctacccaacg 300
ctgccgcctg gcacgggccg ccgcatccac agctaccgag tgtatactct gaaagagcga 360
tgcctccagg ttgtccggag cctagtcaag cctgagaatt acaggagact ggacatcgtc 420
aggtcgctct acgaagatct ggaagaccac ccaaatgtgc agaaagacct ggagcggctg 480
acacaggagc gcattgcaca tcaacggatg ggagatgga 519
<210> 5
<211> 172
<212> PRT
<213> homo sapiens
<400> 5
Met Pro Arg Arg Ala Glu Asn Trp Asp Glu Ala Glu Val Gly Ala Glu
1 5 10 15
Glu Ala Gly Val Glu Glu Tyr Gly Pro Glu Glu Asp Gly Gly Glu Glu
20 25 30
Ser Gly Ala Glu Glu Ser Gly Pro Glu Glu Ser Gly Pro Glu Glu Leu
35 40 45
Gly Ala Glu Glu Glu Met Glu Ala Gly Arg Pro Arg Pro Val Leu Arg
50 55 60
Ser Val Asn Ser Arg Glu Pro Ser Gln Val Ile Phe Cys Asn Arg Ser
65 70 75 80
Pro Arg Val Val Leu Pro Val Trp Leu Asn Phe Asp Gly Glu Pro Gln
85 90 95
Pro Tyr Pro Thr Leu Pro Pro Gly Thr Gly Arg Arg Ile His Ser Tyr
100 105 110
Arg Val Tyr Thr Leu Lys Glu Arg Cys Leu Gln Val Val Arg Ser Leu
115 120 125
Val Lys Pro Glu Asn Tyr Arg Arg Leu Asp Ile Val Arg Ser Leu Tyr
130 135 140
Glu Asp Leu Glu Asp His Pro Asn Val Gln Lys Asp Leu Glu Arg Leu
145 150 155 160
Thr Gln Glu Arg Ile Ala His Gln Arg Met Gly Asp
165 170
<210> 6
<211> 173
<212> PRT
<213> homo sapiens
<400> 6
Met Pro Arg Arg Ala Glu Asn Trp Asp Glu Ala Glu Val Gly Ala Glu
1 5 10 15
Glu Ala Gly Val Glu Glu Tyr Gly Pro Glu Glu Asp Gly Gly Glu Glu
20 25 30
Ser Gly Ala Glu Glu Ser Gly Pro Glu Glu Ser Gly Pro Glu Glu Leu
35 40 45
Gly Ala Glu Glu Glu Met Glu Ala Gly Arg Pro Arg Pro Val Leu Arg
50 55 60
Ser Val Asn Ser Arg Glu Pro Ser Gln Val Ile Phe Cys Asn Arg Ser
65 70 75 80
Pro Arg Val Val Leu Pro Val Trp Leu Asn Phe Asp Gly Glu Pro Gln
85 90 95
Pro Tyr Pro Thr Leu Pro Pro Gly Thr Gly Arg Arg Ile His Ser Tyr
100 105 110
Arg Val Tyr Thr Leu Lys Glu Arg Cys Leu Gln Val Val Arg Ser Leu
115 120 125
Val Lys Pro Glu Asn Tyr Arg Arg Leu Asp Ile Val Arg Ser Leu Tyr
130 135 140
Glu Asp Leu Glu Asp His Pro Asn Val Gln Lys Asp Leu Glu Arg Leu
145 150 155 160
Thr Gln Glu Arg Ile Ala His Gln Arg Met Gly Asp Gly
165 170
<210> 7
<211> 21
<212> DNA
<213> homo sapiens
<400> 7
tactacagag gcatgaacac c 21
<210> 8
<211> 21
<212> DNA
<213> homo sapiens
<400> 8
cagacaagtc accaaccgct a 21

Claims (4)

1. A mutated VHL gene characterized in that at genomic position chr3:10191647, base T is mutated to base G; the reference genomic version is GRCh 37.
2. The mutated VHL gene according to claim 1, characterized in that at genomic position chr3:10191630-3:10191681, the sequence is SEQ ID NO 2.
3. A kit for the detection of a monogenic hypertensive gene, comprising primers for amplifying the mutated VHL gene according to claim 1 or 2.
4. The kit for detecting monogenic hypertension genes according to claim 3, wherein the sequences of the primers are SEQ ID NO. 7 and SEQ ID NO. 8.
CN202111038660.5A 2021-09-06 2021-09-06 Single gene hypertension gene detection kit based on mutated VHL gene Pending CN113684220A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1952171A (en) * 2005-10-20 2007-04-25 上海市高血压研究所 Gene detection chip for determining pheochromocytoma-related gene and application thereof
CN109750101A (en) * 2019-02-15 2019-05-14 中国医学科学院阜外医院 Detect gene panel and its application of monogenic inheritance hypertension

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1952171A (en) * 2005-10-20 2007-04-25 上海市高血压研究所 Gene detection chip for determining pheochromocytoma-related gene and application thereof
CN109750101A (en) * 2019-02-15 2019-05-14 中国医学科学院阜外医院 Detect gene panel and its application of monogenic inheritance hypertension

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALEXANDER WEBER等: "Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma", 《HORM CANCER》 *
HILDE DANNENBERG等: "Von Hippel-Lindau gene alterations in sporadic benign and malignant pheochromocytomas", 《INT J CANCER》 *
MALACHIGRIFFITH: "MyVariant Identifiers:chr3:g.10191647T>G（hg19），CIViC:CA351756720", 《CIVIC》 *

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Application publication date: 20211123